Generation of HaCaT cell line that continually express the receptor interacting serine-threonine kinase (RİPK4)

The multilayered epidermis acts as a protective barrier of the skin. Upon dermis originated differentiation signal, basal layer localized stem cells start to divide asymmetrically and the daughter cells that move apart from stem cells enters differentiation process to form epidermal suprabasal layers. This process, known as epidermal differentiation, is regulated very precisely and failures can cause congenital anomalies. Receptor Interacting Serine Threonine Kinase 4 (RIPK4) is one of the leading proteins that regulate epidermal differentiation. Regarding its function, RIPK4 variations have been associated with congenital ectodermal development defects such as Bartsocas-Papas, Popliteal pytergium, AEC-Like and CHAND syndromes. In laboratory conditions in which keratinocyte cell lines are used, the epidermal differentiation process is between two and three weeks. In studies investigating epidermal differentiation by laboratory analysis, HaCaT cells, which can grow in simple conditions and easy to handle, are generally used instead of normal human keratinocytes (NHC). However, the transfection efficiency of the gene of interest to HaCaT cells is very low. In addition, in these transfected cells the expression of the gene of interest can last only couple days. However, in epidermal differentiation-based studies the expression of the gene of interest should last for at least 14 days. Therefore, the aim of this thesis is to establish transgenic HaCaT cells that allow stable integration of gene of interest into their genome and to over express RIPK4 continuously by using these established HaCaT cells. Within this scope firstly FRT cassette, which allows the integration of gene of interest into the HaCaT cells genome, was integrated to the genome of HaCaT cells genome by using Flp-InTM system. The integration of single copy FRT cassette to the transcriptionally active site of the genome without disturbing the vital cellular functions was confirmed. After that, RIPK4 was integrated to the HaCaT cell genome through FRT cassette to provide continuous expression of RIPK4. With these transgenic cells, it will be possible to design long-term experiments such as epidermal differentiation. Keyword: Epidermal differentiation, Flp-In system, HaCaT, Stable transfection, RIPK4